The one or two osteoporosis drugs now available that build new bone rather than simply prevent further bone loss are not without issues, and the market for anabolic therapy remains open. Progenra, a ubiquitin based drug discovery company, is currently seeking such a therapy for treatment of osteoporosis. The ubiquitin pathway provides several targets for the discovery of selective therapeutic agents, including E3 ligases, which attach ubiquitin to target proteins, marking them for degradation in the proteasome. The ubiquitin E3 ligase Praja1 ubiquitylates and causes degradation of the transcriptional coactivator MAGE-D1/Dlxin-1 (for the transcription factors Dlx5 and Dlx3), interfering with the transcription of an osteogenic gene program (including Runx2, OC, and BSP). The end result of Praja1 inhibition is thus bone formation. Progenra is screening small molecule and natural product-containing libraries for inhibitors of this ligase for development as anabolic agents to treat osteoporosis. Using existing in vitro screening assays, Progenra is in the process of identifying specific inhibitors of the E3 ligase activity of Praja1. However, such potential therapeutics must next be evaluated in cell-based functional assays, and the best of these further evaluated in animal models in the course of lead optimization and preclinical development/clinical candidate selection. In the present application, cell-based in vitro assays are proposed wherein the functional activity of lead compounds from Praja1 inhibitor screens will be evaluated. The assays will assess the effects of candidate compounds on cells at both the transcriptional and bone-formation levels and will be validated by the use of Praja1 siRNA and generic proteasome inhibitors. The work will be accomplished in three aims. First, an assay will be established to evaluate Dlx2-, 3-, and 5-mediated transcription and activation of Runx2 and osterix, using cultured cell lines. Next, a model for osteoblast differentiation and function will be established. Finally, the in vitro models developed will be tested using a battery of proteasome inhibitors to mimick preservation of the proteins marked for degradation by active Praja1. In Phase II, appropriate compounds that have been found positive in these cell-based functional assays will be tested in animal models of bone formation. PUBLIC HEALTH RELEVANCE: Treating the debilitating bone disease osteoporosis with drugs that cause new bone growth, rather than merely prevent further bone loss, remains a goal of many pharmaceutical efforts. Today only one or two drugs commonly prescribed for osteoporosis work by this mechanism; most inhibit further bone loss. Progenra has created a screening technology that will allow it to search large compound collections to find inhibitors of an enzyme called Praja-1, which is responsible for blocking new bone formation. Such inhibitors have the potential to permit new bone formation in patients with osteoporosis. Before an inhibitor found in the screen can be considered for clinical development, it must be tested to see whether it acts as predicted in cells and then in whole animals. This proposal has to do with the cellular evaluation of Praja1 inhibitors found in screening. It is proposed here to construct assays using cultured cells that will measure the ability of a compound (a potent Praja1 inhibitor from the screen) to allow the expression of genes normally blocked by active Praja1, and to promote differentiation in mouse cells that is consistent with new bone formation. All compounds under consideration for preclinical will be tested in these cell-based functional assays. [unreadable] [unreadable] [unreadable]